Antibodies for inhibiting blood coagulation and methods of use thereof

ABSTRACT

The invention includes antibodies that provide superior anti-coagulant activity by binding native human TF with high affinity and specificity. Antibodies of the invention can effectively inhibit blood coagulation in vivo. Antibodies of the invention can bind native human TF, either alone or present in a TF:VIIa complex, effectively preventing factor X binding to TF or that complex, and thereby reducing blood coagulation. Preferred antibodies of the invention specifically bind a conformational epitope predominant to native human TF, which epitope provides an unexpectedly strong antibody binding site.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to novel antibodies and methods ofusing the antibodies to inhibit blood coagulation. In particular, theinvention relates to novel antibodies that can specifically bind nativehuman tissue factor with high affinity. The antibodies of the inventionare useful for a variety of applications, particularly for reducingblood coagulation in vivo.

[0003] 2. Background

[0004] Blood clotting assists homeostasis by minimizing blood loss.Generally, blood clotting requires vessel damage, platelet aggregation,coagulation factors and inhibition of fibrinolysis. The coagulationfactors act through a cascade that relates the vessel damage toformation of a blood clot (see generally L. Stryer, Biochemistry, 3rdEd, W. H. Freeman Co., New York; and A. G. Gilman et al., ThePharmacological Basis of Therapeutics, 8th Edition, McGraw Hill Inc.,New York, pp. 1311-1331).

[0005] There is general agreement that factor X (FX) activation tofactor Xa (FXa) is a critical step in the blood coagulation process.Generally, FX is converted to FXa by binding a catalytically activecomplex that includes “tissue factor” (TF). TF is acontrollably-expressed cell membrane protein that binds factor VII/VIIato produce the catalytically active complex (TF:VIIa). A blood clotfollows FXa-mediated activation of prothrombin. Blood clotting can beminimized by inactivation of TF to non-native forms which cannotoptimally produce the TF:VIIa complex. Excessive formation of FXa isbelieved to contribute to various thromboses including restenosis.

[0006] Thrombosis may be associated with invasive medical proceduressuch as cardiac surgery (e.g. angioplasty), abdominothoracic surgery,arterial surgery, deployment of an implementation (e.g., a stent orcatheter), or endarterectomy. Further, thrombosis may accompany variousthromboembolic disorders and coagulopathies such as a pulmonary embolism(e.g., atrial fibrillation with embolization) and disseminatedintravascular coagulation, respectively. Manipulation of body fluids canalso result in an undesirable thrombus, particularly in bloodtransfusions or fluid sampling, as well as procedures involvingextracorporeal circulation (e.g., cardiopulmonary bypass surgery) anddialysis.

[0007] Anti-coagulants are frequently used to alleviate or avoid bloodclots associated with thrombosis. Blood clotting often can be minimizedor eliminated by administering a suitable anti-coagulant or mixturethereof, including one or more of a coumarin derivative (e.g., warfinand dicumarol) or a charged polymer (e.g., heparin, hirudin or hirulog).See e.g., Gilman et al., supra, R. J. Beigering et al., Ann. Hemathol.,72:177 (1996); J. D. Willerson, Circulation, 94:866 (1996).

[0008] However, use of anti-coagulants is often associated with sideeffects such as hemorrhaging, re-occlusion, “white-clot” syndrome,irritation, birth defects, thrombocytopenia and hepatic dysfunction.Long-term administration of anti-coagulants can particularly increaserisk of life-threatening illness (see e.g., Gilman et al., supra).

[0009] Certain antibodies with anti-platelet activity have also beenused to alleviate various thromboses. For example, ReoPro™ is atherapeutic antibody that is routinely administered to alleviate variousthromboembolic disorders such as those arising from angioplasty,myocardial infarction, unstable angina and coronary artery stenoses.Additionally, ReoPro™ can be used as a prophylactic to reduce the riskof myocardial infarction and angina (J. T. Willerson, Circulation,94:866 (1996); M. L. Simmons et al., Circulation, 89:596 (1994)).

[0010] Certain anti-coagulant antibodies are also known. Particularly,certain TF-binding antibodies have been reported to inhibit bloodcoagulation, presumably by interfering with assembly of a catalyticallyactive TF:VIIa complex (see e.g., Jeske et al., SEM in THROM. and HEMO,22:213 (1996); Ragni et al., Circulation, 93:1913 (1996); EuropeanPatent No. 0 420 937 B1; W. Ruf et al., Throm. Haemosp., 66:529 (1991);M. M. Fiorie et al., Blood, 8:3127 (1992)).

[0011] However, current TF-binding antibodies exhibit significantdisadvantages which can minimize their suitably as anti-coagulants. Forexample, current TF-binding antibodies do not exhibit sufficient bindingaffinity for optimal anti-coagulant activity. Accordingly, for manythrombotic conditions, to compensate for such ineffective bindingaffinities, unacceptably high antibody levels must be administered tominimize blood coagulation. Further, current TF-binding antibodies donot effectively discriminate between native TF and non-native forms ofTF, i.e. the current antibodies do not exhibit sufficient bindingspecificity. Still further, current TF-binding antibodies can notprevent FX from binding to TF and/or TF:VIIa complex.

[0012] It would thus be desirable to have an anti-coagulant antibodythat binds native human TF with high affinity and selectivity to therebyinhibit undesired blood coagulation and the formation of blood clots. Itwould be further desirable to have such an anti-coagulant antibody thatprevents the binding of Factor X to TF/VIIa complex.

SUMMARY OF THE INVENTION

[0013] We have now discovered antibodies that provide superioranti-coagulant activity by binding native human TF with high affinityand specificity. Antibodies of the invention can effectively inhibitblood coagulation in vivo. Antibodies of the invention can bind nativehuman TF, either alone or present in a TF:VIIa complex, effectivelypreventing factor X binding to TF or that complex, and thereby reducingblood coagulation.

[0014] Preferred antibodies of the invention are monoclonal andspecifically bind a conformational epitope predominant to native humanTF, which epitope provides an unexpectedly strong antibody binding site.Indeed, preferred antibodies of the invention bind to native human TF atleast about 5 times greater, more typically at least about ten timesgreater than the binding affinity exhibited by prior anti-coagulantantibodies. Additionally, preferred antibodies of the invention areselective for native human TF, and do not substantially bind non-nativeor denatured TF. H36.D2.B7 (secreted by hybridoma ATCC HB-12255) is anespecially preferred antibody of the invention.

[0015] Preferred antibodies of the invention bind TF so that FX does noteffectively bind to the TF/factor VIIa complex whereby FX is noteffectively converted to its activated form (FXa). Preferred antibodiesof the invention can inhibit TF function by effectively blocking FXbinding or access to TF molecules. See, for instance, the results ofExample 3 which follows.

[0016] Preferred antibodies of the invention also do not significantlyinhibit the interaction or binding between TF and factor VIIa, orinhibit activity of a TF:factor VIIa complex with respect to materialsother than FX. See, for instance, the results of Example 4 whichfollows.

[0017] The invention also provides nucleic acids that encode antibodiesof the invention. Nucleic acid and amino acid sequences (SEQ ID:NOS 1-4)of variable regions of H36.D2.B7 are set forth in FIGS. 1A and 1B of thedrawings.

[0018] In preferred aspects, the invention provides methods forinhibiting blood coagulation and blood clot formation, and methods forreducing human TF levels.

[0019] In general, antibodies of the invention will be useful tomodulate virtually any biological response mediated by FX binding to TFor the TF:VIIa complex, including blood coagulation as discussed above,inflammation and other disorders.

[0020] Antibodies of the invention are particularly useful to alleviatevarious thromboses, particularly to prevent or inhibit restenosis, orother thromboses following an invasive medical procedure such asarterial or cardiac surgery (e.g., angioplasty). Antibodies of theinvention also can be employed to reduce or even effectively eliminateblood coagulation arising from use of medical implementation (e.g., acatheter, stent or other medical device). Preferred antibodies of theinvention will be compatible with many anti-coagulant, anti-platelet andthrombolytic compositions, thereby allowing administration in a cocktailformat to boost or prolong inhibition of blood coagulation.

[0021] Antibodies of the invention also can be employed as ananti-coagulant in extracorporeal circulation of a mammal, particularly ahuman subject. In such methods, one or more antibodies of the inventionis administered to the mammal in an amount sufficient to inhibit bloodcoagulation prior to or during extracorporeal circulation such as may beoccur with cardiopulmonary bypass surgery, organ transplant surgery orother prolonged surgeries.

[0022] Antibodies of the invention also can be used as a carrier fordrugs, particularly pharmaceuticals targeted for interaction with ablood clot such as strepokinase, tissue plasminogen activator (t-PA) orurokinase. Similarly, antibodies of the invention can be used as acytotoxic agent by conjugating a suitable toxin to the antibody.Conjugates of antibodies of the invention also can be used to reducetissue factor levels in a mammal, particularly a human, by administeringto the mammal an effective amount of an antibody of the invention whichis covalently linked a cell toxin or an effector molecule to providecomplement-fixing ability and antibody-dependent cell-mediatedcytotoxicity, whereby the antibody conjugate contacts cells expressingtissue factor to thereby reduce tissue factor levels in the mammal.

[0023] Antibodies of the invention also can be employed in in vivodiagnostic methods including in vivo diagnostic imaging of native humanTF.

[0024] Antibodies of the invention also can be used in in vitro assaysto detect native TF in a biological sample including a body fluid (e.g.,plasma or serum) or tissue (e.g., a biopsy sample). More particularly,various heterogeneous and homogeneous immunoassays can be employed in acompetitive or non-competitive format to detect the presence andpreferably an amount of native TF in the biological sample.

[0025] Such assays of the invention are highly useful to determine thepresence or likelihood of a patient having a blood coagulation or ablood clot. That is, blood coagulation is usually accompanied by TFexpression on cells surfaces such as cells lining the vasculature. Inthe absence of blood coagulation, TF is not usually expressed. Thus, thedetection of TF in a body fluid sample by an assay of the invention willbe indicative of blood coagulation.

[0026] Antibodies of the invention also can be used to preparesubstantially pure native TF, particularly native human TF, from abiological sample. Antibodies of the invention also can be used fordetecting and purifying cells which express native TF.

[0027] Antibodies of the invention also can be employed as a componentof a diagnostic kit, e.g. for detecting and preferably quantitatingnative TF in a biological sample. Other aspects of the invention arediscussed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIGS. 1A and 1B shows the nucleic acid (SEQ ID NOS:1 and 3) andamino acid (SEQ ID NOS:2 and 4) sequences of light chain and heavy chainvariable regions of H36.D2.B7 with hypervariable regions (CDRs orComplementarity Determining Regions) underlined (single underline fornucleic acid sequences and double underline for amino acid sequences).

[0029]FIG. 2 shows association (K_(a)) and disassociation (K_(d))constants of anti-tissue factor antibodies as determined by ELISA orBIACore analysis.

[0030]FIG. 3 shows inhibition of TF:VIIa complex mediated FX activationby pre-incubation with anti-tissue factor antibodies.

[0031]FIG. 4 shows inhibition of TF/VIIa activity toward theFVIIa-specific substrate S-2288 by anti-tissue factor antibodies.

[0032]FIG. 5 shows the capacity of the H36 antibody to increaseprothrombin time (PT) in a TF-initiated coagulation assay.

[0033]FIGS. 6A and 6B graphically show the relationship between FXaformation and molar ratio of the H36.D2 antibody and rHTF. FIG. 6A:H36.D2 was pre-incubated with the FT:VIIa complex prior to adding FX.FIG. 6B: H36.D2, TF:VIIa and FX were added simultaneously.

[0034]FIG. 7 shows inhibition of TF:VIIa activity by the H36.D2 antibodyin a J-82 cell activation assay.

[0035]FIGS. 8A and 8B are representations of dot blots showing that theH36.D2 antibody binds a conformational epitope on rhTF. Lane 1-nativerHTF, Lane 2-native rhTF treated with 8M urea, Lane 3-native rHTFtreated with 8M urea and 5 mM DTT. In FIG. 8A, the blot was exposed forapproximately 40 seconds, whereas in FIG. 8B, the blot was exposed for120 seconds.

DETAILED DESCRIPTION OF THE INVENTION

[0036] As discussed above, preferred antibodies of the invention exhibitsubstantial affinity for native human TF. In particular, preferredantibodies of the invention exhibit an association constant (K_(a), M⁻¹)for native human TF of at least about 1×10⁸ as determined by surfaceplasmon analysis (particularly, BIACore analysis in accordance with theprocedures of Example 1 which follows), more preferably at least about5×10⁸ as determined by surface plasmon analysis, still more preferably aK_(a) (K_(a), M⁻¹) for native human TF of at least about 1×10¹⁰ asdetermined by surface plasmon analysis. Such substantial bindingaffinity of antibodies of the invention contrast sharply from much lowerbinding affinities of previously reported antibodies.

[0037] In this regard, a quite low of effective concentration of anantibody of the invention can be employed, e.g. a relatively lowconcentration of antibody can be employed to inhibit TF function asdesired (e.g. at least about 95, 98 or 99 percent inhibition) in an invitro assay such as described in Example 3 which follows.

[0038] The preferred antibodies are also highly specific for nativehuman TF, and preferably do not substantially bind with non-native TF.Preferred antibodies do not substantially bind non-native TF or otherimmunologically unrelated molecules as determined, e.g. by standard dotblot assay (e.g. no or essentially no binding to non-native TF visuallydetected by such dot blot assay). References herein to “non-native TF”mean a naturally-occurring or recombinant human TF that has been treatedwith a choatropic agent so that the TF is denatured. Typical choatropicagents include a detergent (e.g. SDS), urea combined with dithiothreotolor β-mercaptoethanol; guanidine hydrochloride and the like. The H36,H36.D2 or H36.D2.B7 antibody does not substantially bind to suchnon-native TF. See, for instance, the results of Example 8 which followsand is a dot blot assay.

[0039] As discussed above, preferred antibodies of the invention alsobind with TF so that FX does not effectively bind to the TF/factor VIIacomplex whereby FX is not effectively converted to its activated form(FXa). Particularly preferred antibodies of the invention exhibit willstrongly inhibit FX activity to a TF/factor VIIa complex, e.g. aninhibition of at least about 50%, more preferably at least about 80%,and even more preferably at least about 90% or 95%, even at low TFconcentrations such as less than about 1.0 nM TF, or even less thanabout 0.20 nM or 0.10 nM TF, as determined by a standard in vitrobinding assay such as that of Example 3 which follows and includescontacting FX with a TF:factor VIIa complex both in the presence (i.e.experimental sample) and absence (i.e. control sample) of an antibody ofthe invention and determining the percent difference of conversion of FXto FXa between the experimental and control samples.

[0040] Antibodies of the invention are preferably substantially purewhen used in the disclosed methods and assays. References to an antibodybeing “substantially pure” mean an antibody or protein which has beenseparated from components which naturally accompany it. For example, byusing standard immunoaffinity or protein A affinity purificationtechniques, an antibody of the invention can be purified from ahybridoma culture by using native TF as an antigen or protein A resin.Similarly, native TF can be obtained in substantially pure form by usingan antibody of the invention with standard immunoaffinity purificationtechniques. Particularly, an antibody or protein is substantially purewhen at least 50% of the total protein (weight % of total protein in agiven sample) is an antibody or protein of the invention. Preferably theantibody or protein is at least 60 weight % of the total protein, morepreferably at least 75 weight %, even more preferably at least 90 weight%, and most preferably at least 98 weight % of the total material.Purity can be readily assayed by known methods such as SDS (PAGE) gelelectrophoresis, column chromatography (e.g., affinity chromatography)or HPLC analysis.

[0041] The nucleic acid (SEQ ID NOS: 1 and 3) and amino acid (SEQ IDNOS: 2 and 4) sequences of a preferred antibody of the invention(H36.D2.B7) are shown in FIGS. 1A and 1B of the drawings. SEQ ID NOS. 1and 2 are the nucleic acid and amino acid respectively of the lightchain variable region, and SEQ ID NOS. 3 and 4 are the nucleic acid andamino acid respectively of the heavy chain variable region, withhypervariable regions (CDRs or Complementarity Determining Regions)underlined in all of those sequences.

[0042] Additional preferred antibodies of the invention will havesubstantial sequence identity to either one or both of the light chainor heavy sequences shown in FIGS. 1A and 1B. More particularly,preferred antibodies include those that have at least about 70 percenthomology (sequence identity) to SEQ ID NOS. 2 and/or 4, more preferablyabout 80 percent or more homology to SEQ ID NOS. 2 and/or 4, still morepreferably about 85, 90 or 95 percent or more homology to SEQ ID NOS. 2and/or 4.

[0043] Preferred antibodies of the invention will have high sequenceidentity to hypervariable regions (shown with double underlining inFIGS. 1A and 1B) of SEQ ID NOS. 2 and 4). Especially preferredantibodies of the invention will have one, two or three hypervariableregions of a light chain variable region that have high sequenceidentity (at least 90% or 95% sequence identity) to or be the same asone, two or three of the corresponding hypervariable regions of thelight chain variable region of H36.D2.B7 (those hypervariable regionsshown with underlining in FIG. 1A and are the following: 1) LASQTID (SEQID NO:5); 2) AATNLAD (SEQ ID NO:6); and 3) QQVYSSPFT (SEQ ID NO:7)).

[0044] Especially preferred antibodies of the invention also will haveone, two or three hypervariable regions of a heavy chain variable regionthat have high sequence identity (at least 90% or 95% sequence identity)to or be the same as one, two or three of the correspondinghypervariable regions of the heavy chain variable region of H36.D2.B7(those hypervariable regions shown with underlining in FIG. 1B and arethe following: 1) TDYNVY (SEQ ID NO:8); 2) YIDPYNGITIYDQNFKG (SEQ IDNO:9); and 3) DVTTALDF (SEQ ID NO:10).

[0045] Nucleic acids of the invention preferably are of a lengthsufficient (preferably at least about 100, 200 or 250 base pairs) tobind to the sequence of SEQ ID NO:1 and/or SEQ ID NO:3 under thefollowing moderately stringent conditions (referred to herein as “normalstringency” conditions): use of a hybridization buffer comprising 20%formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at atemperature of 37° C. and remaining bound when subject to washing oncewith that SSC buffer at 37° C.

[0046] More preferably, nucleic acids of the invention (preferably atleast about 100, 200 or 250 base pairs) will bind to the sequence of SEQID NO:1 and/or SEQ ID NO:3 under the following highly stringentconditions (referred to herein as “high stringency” conditions): use ofa hybridization buffer comprising 20% formamide in 0.9M saline/0.09Msodium citrate (SSC) buffer at a temperature of 42° C. and remainingbound when subject to washing twice with that SSC buffer at 42° C.

[0047] Nucleic acids of the invention preferably comprise at least 20base pairs, more preferably at least about 50 base pairs, and still morepreferably a nucleic acid of the invention comprises at least about 100,200, 250 or 300 base pairs.

[0048] Generally preferred nucleic acids of the invention will expressan antibody of the invention that exhibits the preferred bindingaffinities and other properties as disclosed herein.

[0049] Preferred nucleic acids of the invention also will havesubstantial sequence identity to either one or both of the light chainor heavy sequences shown in FIGS. 1A and 1B. More particularly,preferred nucleic acids will comprise a sequence that has at least about70 percent homology (sequence identity) to SEQ ID NOS. 1 and/or 3, morepreferably about 80 percent or more homology to SEQ ID NOS. 1 and/or 3,still more preferably about 85, 90 or 95 percent or more homology to SEQID NOS. 1 and/or 3.

[0050] Particularly preferred nucleic acid sequences of the inventionwill have high sequence identity to hypervariable regions (shown withunderlining in FIGS. 1A and 1B) of SEQ ID NOS. 1 and 3). Especiallypreferred nucleic acids include those that code for an antibody lightchain variable region and have one, two or three sequences that code forhypervariable regions and have high sequence identity (at least 90% or95% sequence identity) to or be the same as one, two or three of thesequences coding for corresponding hypervariable regions of H36.D2.B7(those hypervariable regions shown with underlining in FIG. 1A and arethe following: 1) CTGGCAAGTCAGACCATTGAT (SEQ ID NO:11); 2) GCTGCCACCAACTTGGCAGAT (SEQ ID NO:12); and 3) CAACAAGTTTACAGTTCT CCATTCACGT (SEQID NO:13)).

[0051] Especially preferred nucleic acids also code for an antibodyheavy chain variable region and have one, two or three sequences thatcode for hypervariable regions and have high sequence identity (at least90% or 95% sequence identity) to or be the same as one, two or three ofthe sequences coding for corresponding hypervariable regions ofH36.D2.B7 (those hypervariable regions shown with underlining in FIG. 1Band are the following: 1) ACTGACTACAACGTGTAC (SEQ ID NO: 14); 2)TATATTGAT CCTTACAATGGTATTACTATCTACGACCAGAACTTCAAGGGC (SEQ ID NO:15); and3) GATGTGACTACGGCCCTTGACTTC (SEQ ID NO:16)).

[0052] Nucleic acids of the invention are isolated, usually constitutesat least about 0.5%, preferably at least about 2%, and more preferablyat least about 5% by weight of total nucleic acid present in a givenfraction. A partially pure nucleic acid constitutes at least about 10%,preferably at least about 30%, and more preferably at least about 60% byweight of total nucleic acid present in a given fraction. A pure nucleicacid constitutes at least about 80%, preferably at least about 90%, andmore preferably at least about 95% by weight of total nucleic acidpresent in a given fraction.

[0053] Antibodies of the invention can be prepared by techniquesgenerally known in the art, and are typically generated to a purifiedsample of native TF, typically native human TF, preferably purifiedrecombinant human tissue factor (rhTF). Truncated recombinant humantissue factor or “rhTF” (composed of 243 amino acids and lacking thecytoplasmic domain) is particularly preferred to generate antibodies ofthe invention. The antibodies also can be generated from an immunogenicpeptide that comprises one or more epitopes of native TF that are notexhibited by non-native TF. References herein to “native TF” includesuch TF samples, including such rhTF. As discussed above, monoclonalantibodies are generally preferred, although polyclonal antibodies alsocan be employed.

[0054] More particularly, antibodies can be prepared by immunizing amammal with a purified sample of native human TF, or an immunogenicpeptide as discussed above, alone or complexed with a carrier. Suitablemammals include typical laboratory animals such as sheep, goats,rabbits, guinea pigs, rats and mice. Rats and mice, especially mice, arepreferred for obtaining monoclonal antibodies. The antigen can beadministered to the mammal by any of a number of suitable routes such assubcutaneous, intraperitoneal, intravenous, intramuscular orintracutaneous injection. The optimal immunizing interval, immunizingdose, etc. can vary within relatively wide ranges and can be determinedempirically based on this disclosure. Typical procedures involveinjection of the antigen several times over a number of months.Antibodies are collected from serum of the immunized animal by standardtechniques and screened to find antibodies specific for native human TF.Monoclonal antibodies can be produced in cells which produce antibodiesand those cells used to generate monoclonal antibodies by using standardfusion techniques for forming hybridoma cells. See G. Kohler, et al.,Nature, 256:456 (1975). Typically this involves fusing an antibodyproducing cell with an immortal cell line such as a myeloma cell toproduce the hybrid cell. Alternatively, monoclonal antibodies can beproduced from cells by the method of Huse, et al., Science, 256:1275(1989).

[0055] One suitable protocol provides for intraperitoneal immunizationof a mouse with a composition comprising purified rhTF complex conductedover a period of about two to seven months. Spleen cells then can beremoved from the immunized mouse. Sera from the immunized mouse isassayed for titers of antibodies specific for rhTF prior to excision ofspleen cells. The excised mouse spleen cells are then fused to anappropriate homogenic or heterogenic (preferably homogenic) lymphoidcell line having a marker such as hypoxanthine-guaninephosphoribosyltransferase deficiency (HGPRT⁻) or thymidine kinasedeficiency (TK⁻). Preferably a myeloma cell is employed as the lymphoidcell line. Myeloma cells and spleen cells are mixed together, e.g. at aratio of about 1 to 4 myeloma cells to spleen cells. The cells can befused by the polyethylene glycol (PEG) method. See G. Kohler, et al.,Nature, supra. The thus cloned hybridoma is grown in a culture medium,e.g. RPMI-1640. See G. E. More, et al., Journal of American MedicalAssociation, 199:549 (1967). Hybridomas, grown after the fusionprocedure, are screened such as by radioimmunoassay or enzymeimmunoassay for secretion of antibodies that bind specifically to thepurified rhTF, e.g. antibodies are selected that bind to the purifiedrhTF, but not to non-native TF. Preferably an ELISA is employed for thescreen. Hybridomas that show positive results upon such screening can beexpanded and cloned by limiting dilution method. Further screens arepreferably performed to select antibodies that can bind to rhTF insolution as well as in a human fluid sample. The isolated antibodies canbe further purified by any suitable immunological technique includingaffinity chromatography. A hybridoma culture producing the particularpreferred H36.D2.B7 antibody has been deposited pursuant to the BudapestTreaty with the American Type Culture Collection (ATCC) at 12301Parklawn Drive, Rockville, Md., 10852. The hybridoma culture wasdeposited with the ATCC on Jan. 8, 1997 and was assigned AccessionNumber ATCC HB-12255.

[0056] For human therapeutic applications, it may be desirable toproduce chimeric antibody derivatives, e.g. antibody molecules thatcombine a non-human animal variable region and a human constant region,to thereby render the antibodies less immunogenic in a human subjectthan the corresponding non-chimeric antibody. A variety of types of suchchimeric antibodies can be prepared, including e.g. by producing humanvariable region chimeras, in which parts of the variable regions,especially conserved regions of the antigen-binding domain, are of humanorigin and only the hypervariable regions are of non-human origin. Seealso discussions of humanized chimeric antibodies and methods ofproducing same in S. L. Morrison, Science, 229:1202-1207 (1985); Oi etal., BioTechniques, 4:214 (1986); Teng et al., Proc. Natl. Acad. Sci.U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today, 4:7279(9183); Olsson et al., Meth. Enzymol., 9:3-16 (1982). Additionally,transgenic mice can be employed. For example, transgenic mice carryinghuman antibody repertoires have been created which can be immunized withnative human TF. Splenocytes from such immunized transgenic mice canthen be used to create hybridomas that secrete human monoclonalantibodies that specifically react with native human TF as describedabove. See N. Lonberg et al., Nature, 368:856-859 (1994); L. L. Green etal., Nature Genet., 7:13-21 (1994); S. L. Morrison, Proc. Natl. Acad.Sci. U.S.A., 81:6851-6855 (1994).

[0057] Nucleic acids of antibodies of the invention also can be preparedby polymerase chain reaction (see primers disclosed in Example 1 whichfollows). See generally, Sambrook et al., Molecular Cloning (2d ed.1989). Such nucleic acids also can be synthesized by known methods, e.g.the phosphate triester method (see Oligonucleotide Synthesis, IRL Press(M. J. Gait, ed., 1984)), or by using a commercially available automatedoligonucleotide synthesizer. Such a prepared nucleic acid of theinvention can be employed to express an antibody of the invention byknown techniques. For example, a nucleic acid coding for an antibody ofthe invention can be incorporated into a suitable vector by knownmethods such as by use of restriction enzymes to make cuts in the vectorfor insertion of the construct followed by ligation. The vectorcontaining the inserted nucleic acid sequence, suitably operably linkedto a promoter sequence, is then introduced into host cells forexpression. See, generally, Sambrook et al., supra. Selection ofsuitable vectors can be made empirically based on factors relating tothe cloning protocol. For example, the vector should be compatible with,and have the proper replicon for the host cell that is employed.Further, the vector must be able to accommodate the inserted nucleicacid sequence. Suitable host cells will include a wide variety ofeukaryotic or prokaryotic cells such as E. coli and the like.

[0058] The molecular weight of the antibodies of the invention will varydepending on several factors such as the intended use and whether theantibody includes a conjugated or recombinantly fused toxin,pharmaceutical, or detectable label or the like. In general, an antibodyof the invention will have a molecular weight of between approximately20 to 150 kDa. Such molecular weights can be readily are determined bymolecular sizing methods such as SDS-PAGE gel electrophoresis followedby protein staining or Western blot analysis.

[0059] “Antibody of the invention” or other similar term refers to wholeimmunoglobulin as well immunologically active fragments which bindnative TF. The immunoglobulins and immunologically active fragmentsthereof include an antibody binding site (i.e., peritope capable ofspecifically binding native human TF). Exemplary antibody fragmentsinclude, for example, Fab, F(v), Fab′, F(ab′)₂ fragments, “halfmolecules” derived by reducing the disulfide bonds of immunoglobulins,single chain immunoglobulins, or other suitable antigen bindingfragments (see e.g., Bird et al., Science, pp. 242-424 (1988); Huston etal., PNAS, (USA), 85:5879 (1988); Webber et al., Mol. Immunol., 32:249(1995)). The antibody or immunologically active fragment thereof may beof animal (e.g., a rodent such as a mouse or a rat), or chimeric form(see Morrison et al., PNAS, 81:6851 (1984); Jones et al., Nature, pp.321, 522 (1986)). Single chain antibodies of the invention can bepreferred.

[0060] Similarly, a “nucleic acid of the invention” refers to a sequencewhich can be expressed to provide an antibody of the invention as suchterm is specified to mean immediately above.

[0061] As discussed above, antibodies of the invention can beadministered to a mammal, preferably a primate such as a human, toprevent or reduce thromboses such as restenosis, typically in acomposition including one or more pharmaceutically acceptable non-toxiccarriers such as sterile water or saline, glycols such as polyethyleneglycol, oils of vegetable origin, and the like. In particular,biocompatible, biodegradable lactide polymer, lactide glycolidecopolymer or polyoxyethylene, polyoxypropylene copolymers may be usefulexcipients to control the release of the antibody-containingcompositions described herein. Other potentially useful administrationsystems include ethylene vinyl acetate copolymer particles, osmoticpumps, and implantable infusion systems and liposomes. Generally, ananti-coagulant composition of the invention will be in the form of asolution or suspension, and will preferably include approximately 0.01%to 10% (w/w) of the antibody of the present invention, preferablyapproximately 0.01% to 5% (w/w) of the antibody. The antibody can beadministered as a sole active ingredient in the composition, or as acocktail including one or more other anti-coagulant (e.g., heparin,hirudin, or hirulog), anti-platelet (e.g., ReoPro), or thrombolyticagents (e.g., tissue plasminogen activator, strepokinase and urokinase).Additionally, antibodies of the invention can be administered prior to,or after administration of one or more suitable anti-coagulant,anti-platelet or thrombolytic agents to boost or prolong desiredanti-coagulation activity.

[0062] As also discussed above, antibodies of the invention can beemployed to reduce potential blood coagulation arising from use ofmedical implementation, e.g. an indwelling device such as a catheter,stent, etc. In one preferred method, the implementation can be treatedwith an antibody of the invention (e.g., as a 1 mg/ml saline solution)prior to contact with a body fluid. Alternatively, or in addition, anantibody of the invention can be combined with the body fluid in anamount sufficient to minimize blood clotting.

[0063] Therapeutic anti-coagulant compositions according to theinvention are suitable for use in parenteral or intravenousadministration, particularly in the form of liquid solutions. Suchcompositions may be conveniently administered in unit dose and may beprepared in accordance with methods known in the pharmaceutical art. SeeRemington's Pharmaceutical Sciences, (Mack Publishing Co., Easton Pa.,(1980)). By the term “unit dose” is meant a therapeutic composition ofthe present invention employed in a physically discrete unit suitable asunitary dosages for a primate such as a human, each unit containing apre-determined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent orcarrier. The unit dose will depend on a variety of factors including thetype and severity of thrombosis to be treated, capacity of the subject'sblood coagulation system to utilize the antibody, and degree ofinhibition or neutralization of FX activation desired. Precise amountsof the antibody to be administered typically will be guided by judgementof the practitioner, however, the unit dose will generally depend on theroute of administration and be in the range of 10 ng/kg body weight to50 mg/kg body weight per day, more typically in the range of 100 ng/kgbody weight to about 10 mg/kg body weight per day. Suitable regimentsfor initial administration in booster shots are also variable but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous or intermittent intravenousinfusions may be made sufficient to maintain concentrations of at leastfrom about 10 nanomolar to 10 micromolar of the antibody in the blood.

[0064] In some instances, it may be desirable to modify the antibody ofthe present invention to impart a desirable biological, chemical orphysical property thereto. More particularly, it may be useful toconjugate (i.e. covalently link) the antibody to a pharmaceutical agent,e.g. a fibrinolytic drug such as t-PA, streptokinase, or urokinase toprovide fibrinolytic activity. Such linkage can be accomplished byseveral methods including use of a linking molecule such as aheterobifunctional protein cross-linking agent, e.g. SPDP, carbodimide,or the like, or by recombinant methods.

[0065] In addition to pharmaceuticals such as a fibrinolytic agent, anantibody of the invention can be conjugated to a toxin of e.g. plant orbacterial origin such as diphtheria toxin (i.e., DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin. Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. The toxin can also be an agent active at cell surfacessuch as phospholipases (e.g., phospholipase C). As another example, thetoxin can be a chemotherapeutic drug such as, e.g., vendesine,vincristine, vinblastin, methotrexate, adriamycin, bleomycin, orcisplatin, or, the toxin can be a radionuclide such as, e.g.,iodine-131, yttrium-90, rhenium-188 or bismuth-212 (see generally,Moskaug et al., J. Biol. Chem., 264:15709 (1989); I. Pastan et al.,Cell, 47:641 (1986); Pastan et al., Recombinant Toxins as NovelTherapeutic Agents, Ann. Rev. Biochem., 61:331 (1992); Chimeric ToxinsOlsnes and Phil, Pharmac. Ther., 25:355 (1982); published PCTApplication No. WO 94/29350; published PCT Application No. WO 94/04689;and U.S. Pat. No. 5,620,939). Also, as discussed above, in addition to atoxin, an antibody of the invention can be conjugated to an effectormolecule (e.g. IgG1 or IgG3) to provide complement-fixing ability andantibody-dependent cell-mediated cytoxicity upon administration to amammal.

[0066] Such an antibody/cytotoxin or effector molecule conjugate can beadministered in a therapeutically effective amount to a mammal,preferably a primate such as a human, where the mammal is known to haveor is suspected of having tumor cells, immune system cells, orendothelia capable of expressing TF. Exemplary of such tumor cells,immune system cells and endothelia include malignancies of the breastand lung, monocytes and vascular endothelia.

[0067] Antibodies of the invention also can be conjugated to a varietyof other pharmaceutical agents in addition to those described above suchas, e.g., drugs, enzymes, hormones, chelating agents capable of bindinga radionuclide, as well as other proteins and polypeptides useful fordiagnosis or treatment of disease. For diagnostic purposes, the antibodyof the present invention can be used either detectably-labelled orunlabelled. For example, a wide variety of labels may be suitablyemployed to detectably-label the antibody, such as radionuclides,fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,ligands such as, e.g., haptens, and the like.

[0068] Diagnostic methods are also provided including in vivo diagnosticimaging [see, e.g., A. K. Abbas, Cellular and Molecular Immunology, pg.328 (W. B. Saunders Co. 1991)]. For most in vivo imaging applications,an antibody of the invention can be detectably-labeled with, e.g., ¹²⁵I,³²P, ⁹⁹Tc, or other detectable tag, and subsequently administered to amammal, particularly a human, for a pre-determined amount of timesufficient to allow the antibody to contact a desired target. Thesubject is then scanned by known procedures such as scintigraphic cameraanalysis to detect binding of the antibody. The analysis could aid inthe diagnosis and treatment of a number of thromboses such as thosespecifically disclosed herein. The method is particularly useful whenemployed in conjunction with cardiac surgery, particularly angioplasty,or other surgical procedure where undesired formation of a blood clotcan occur, to visualize the development or movement of a blood clot.

[0069] Antibodies of the invention also can be used to preparesubstantially pure (e.g., at least about 90% pure, preferably at leastabout 96 or 97% pure) native TF, particularly native human TF from abiological sample. For example, native TF can be obtained as previouslydescribed (see e.g., L. V. M. Rao et al., Thrombosis Res., 56:109(1989)) and purified by admixing the solution with a solid supportcomprising the antibody to form a coupling reaction admixture. Exemplarysolid supports include a wall of a plate such as a microtitre plate, aswell as supports including or consisting of polystyrene,polyvinylchloride, a cross-linked dextran such as Sephadex™ (PharmaciaFine Chemicals), agarose, polystyrene beads (Abbott Laboratories),polyvinyl chloride, polystyrene, polyacrylmide in cross-linked form,nitrocellulose or nylon and the like. The TF can then be isolated fromthe solid support in substantially pure form in accordance with standardimmunological techniques. See generally Harlow and Lane supra andAusubel et al. supra).

[0070] As also discussed above, antibodies of the invention can beemployed to detect native human TF in a biological sample, particularlynative TF associated with a blood clot. Exemplary biological samplesinclude blood plasma, serum, saliva, urine, stool, vaginal secretions,bile, lymph, ocular humors, cerebrospinal fluid, cell culture media, andtissue, particularly vascular tissues such as cardiac tissue. Samplesmay be suitably obtained from a mammal suffering from or suspected ofsuffering from a thrombosis, preferably restenosis, associated with,e.g., an invasive medical procedure such as cardiopulmonary bypasssurgery; a heart ailment such as myocardial infarction, cardiomyopathy,valvular heart disease, unstable angina, or artrial fibrillationassociated with embolization; a coagulopathy including disseminatedintravascular coagulation, deployment of an implementation such as astent or catheter; shock (e.g., septic shock syndrome), vascular trauma,liver disease, heat stroke, malignancies (e.g., pancreatic, ovarian, orsmall lung cell carcinoma), lupus, eclampsia, perivascular occlusivedisease, and renal disease.

[0071] For such assays, an antibody of the invention can bedetectably-labelled with a suitable atom or molecule e.g., radioactiveiodine, tritium, biotin, or reagent capable of generating a detectableproduct such as an anti-iodiotypic antibody attached to an enzyme suchas β-galactosidase or horseradish peroxidase, or a fluorescent tag(e.g., fluorescein or rhodamine) in accordance with known methods. Aftercontacting the biological sample with the detectably-labelled antibody,any unreacted antibody can be separated from the biological sample, thelabel (or product) is detected by conventional immunological methodsincluding antibody capture assay, antibody sandwich assay, RIA, ELISA,immunoprecipitation, immunoabsorption and the like (see Harlow and Lane,supra; Ausubel et al. supra). Any label (or product) in excess of thatdetected in a suitable control sample is indicative of the presence ofnative TF, more particularly a blood clot, in the biological sample. Forexample, antibodies of the invention can be detectably-labelled todetect, and preferably quantitate, native TF in accordance with standardimmunological techniques such as antibody capture assay, ELISA, antibodysandwich assay, RIA, immunoprecipitation, immunoabsorption and the like.In some cases, particularly when a tissue is used, the immunologicaltechnique may include tissue fixation with a reagent known tosubstantially maintain protein conformation (e.g., dilute formaldehyde).See generally, Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, (1989); Harlow and Lane in Antibodies: ALaboratory Manual, CSH Publications, N.Y. (1988).

[0072] Antibodies of the invention also can be used for detecting andpurifying cells which express native TF, including fibroblasts, braincells, immune cells, (e.g., monocytes), epithelia, as well as certainmalignant cells. Preferred methods of detecting and purifying the cellsinclude conventional immunological methods (e.g., flow cytometry methodssuch as FACS, and immunopanning). Substantially pure populations ofcells expressing native TF are useful in clinical and research settings,e.g., to establish such cells as cultured cells for screening TF-bindingantibodies.

[0073] The invention also provides test and diagnostic kits fordetection of native TF, particularly native human TF, in a test sample,especially a body fluid such as blood, plasma, etc., or tissue asdiscussed above. A preferred kit includes a detectably-labelled antibodyof the invention. The diagnostic kit can be used in any acceptableimmunological format such as an ELISA format to detect the presence orquantity of native TF in the biological sample.

[0074] All documents mentioned herein are fully incorporated byreference in their entirety.

[0075] The following non-limiting examples are illustrative of theinvention. In the following examples and elsewhere the antibodies H36and H36.D2 are referred to. Those antibodies are the same antibody asH36.D2.B7, but H36 is derived from the mother clone, and H36.D2 isobtained from the primary clone, whereas H36.D2.B7 is obtained from thesecondary clone. No differences have been observed between those threeclones with respect to ability to inhibit TF or other physicalproperties.

EXAMPLE 1 Preparation and Cloning of Anti-rhTF Monoclonal Antibodies

[0076] Monoclonal antibodies against rhTF were prepared as follows.

[0077] A. Immunization and Boosts

[0078] Five female BALB/c mice were immunized with 10 μg each oflipidated, purified rhTF. The mice were initially sensitizedintraperitoneally using Hunter's Titermax adjuvant. Three final boostswere administered in 0.85% NaCl. Boosts were 2, 5.5, and 6.5 months postinitial sensitization. All boosts were given intraperitoneally, exceptthe first which was subcutaneous. The final boost was given 3 dayspre-fusion and 20 μg was administered.

[0079] B. Fusion of Mouse Spleen Lymphocytes with Mouse Myeloma Cells

[0080] Lymphocytes from the spleen of one rhTF immunized BALB/c mousewas fused to X63-Ag8.653 mouse myeloma cells using PEG 1500. Followingexposure to the PEG, the cells were incubated for one hour in heatinactivated fetal bovine serum at 37° C. The fused cells were thenresuspended in RPMI 1640 and incubated overnight at 37° C. with 10% CO₂.The cells were plated the next day using RPMI 1640 and supplemented withmacrophage culture supernatant.

[0081] C. ELISA Development

[0082] Plates for the ELISA assay were coated with 100 microliters ofrecombinant tissue factor (0.25 μg/ml) in a carbonate based buffer. Allsteps were performed at room temperature. Plates were blocked with BSA,washed, and then the test samples and controls were added.Antigen/antibody binding was detected by incubating the plate with goatanti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and thenusing an ABTS peroxidase substrate system (Kirkegaad and PerryLaboratories). Absorbance were read on an automatic plate reader at awavelength of 405 nm.

[0083] D. Stabilization of rhTF Hybridoma Cell Lines

[0084] Two weeks after fusion, screening of hybridoma colonies byspecific rhTF ELISA was started. Screening for new colonies continuedfor three weeks. The positive clones were tested every one to two weeksfor continued antibody production until fifteen stable clones werefrozen down.

[0085] E. Primary and Secondary Cloning

[0086] Limiting dilution cloning was performed on each of the positivestable hybridomas to obtain primary clones. The cells were thawed, grownin culture for a short period of time, and then diluted from 10cells/well to 0.1 cells/well. Primary clones were tested by anti-rhTFELISA and five to six positive clones were expanded and frozen.

[0087] Secondary clone of anti-rhTF antibody, H36.D2.B7, was obtainedfrom primary clone, H36.D2, prepared and stored in liquid nitrogen asdescribed above. Four different dilutions, 5 cells/well, 2 cells/well, 1cell/well, 0.5 cells/well of the primary clone were prepared in 96-wellsmicrotiter plates to start the secondary cloning. Cells were diluted inIMDM tissue culture media containing the following additives: 20% fetalbovine serum (FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO₃. To determine clones thatsecrete anti-rhTF antibody, supernatants from five individual wells ofthe 0.2 cells/well microtiter plate were withdrawn after two weeks ofgrowth and tested for the presence of anti-rhTF antibody by ELISA assaysas described above. All five clones showed positive results in the ELISAassay, with H36.D2.B7 being the best antibody producer. All five cloneswere adapted and expanded in RPMI media containing the followingadditive: 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO₃, and 0.013 mg/ml ofoxalaacetic acid. H36.D2.B7 was purified by Protein A affinitychromatography from the supernatant of cell culture and was tested forits ability to inhibit TF:VIIa in a FX activation assay. The resultsindicated that H36.D2.B7 had the same inhibition as H36.D2 antibody. Allcells were stored in liquid nitrogen.

[0088] F. Isolation of total RNA from H36.D2.B37

[0089] 269 μg of total RNA was isolated from 2.7×10⁵ H36.D2.B7 hybridomacells. The isolation of total RNA was performed as described in theRNeasy Midi Kits protocol from Qiagen. The RNA sample was stored inwater at −20° C. until needed.

[0090] G. cDNA Synthesis and Cloning of Variable Regions of H36.D2.B7Gene

[0091] To obtain the first strand of cDNA, a reaction mixture containing5 μg of total RNA isolated as above, back primers JS300 (all primers areidentified below) for the heavy chain (HC) and OKA 57 for the lightchain (LC), RNase inhibitor, dNTP's, DTT, and superscript II reversetranscriptase, was prepared and incubated at 42° C. for 1 hour. Thereaction tube is then incubated at 65 ° C. for 15 minutes to stop thetranscription. After cooling down, five units of RNase H was then addedand the reaction was allowed to incubate at 37° C. for 20 minutes. ThecDNA sample was stored at −70° C. until needed.

[0092] PCR (polymerase chain reaction) was conducted separately to clonethe variable regions of both HC and LC of anti-rhTF, H36.D2.B7 from thecDNA made as above (nucleic acid and amino acid sequences of those HCand LC variable regions set forth in FIGS. 1A and 1B). Three rounds ofPCR were conducted. Round 1: PCR was run for 35 cycles at 96° C., 53° C.and 72° C. using front primer JS002 and back primer JS300 for HC. For LCfront primer JS009 and back primer OKA 57 were used and PCR was rune for35 cycles at 96° C., 63° C. and 72° C. Round 2: PCR of both HC and LCwas rune the same as in Round 1 with the exception that pMC-18 was usedfor HC front primer and pMC-15 for LC front primer. Round 3: PCR was runfor 30 cycles at 96° C., 60-65° C. and 72° C. using H36HCF and H36HCRprimers for HC. For LC, PCR was run for 30 cycles at 96° C., 58° C. and72° C. using H36LCF and H36LCR primers.

[0093] The following primers were used for cloning H36.D2.B7 variableregions of HC and LC. OKA 57: 5′-GCACCTCCAGATGTTAACTGCTC-3′ (SEQ IDNO:17) JS300: 5′-GAARTAVCCCTTGACCAGGC-3′ (SEQ ID NO:18) JS009:5′-GGAGGCGGCGGTTCTGACATTGTGMTGWCMCART-3′ (SEQ ID NO:19) JS002:5′-ATTTCAGGCCCAGCCGGCCATGGCCGARGTYCARCTKCARCARYC-3′ (SEQ ID NO:20)pMC-15: 5′-CCCGGGCCACCATGKCCCCWRCTCAGYTYCTKG-3′ (SEQ ID NO:21) pMC-18:5′-CCCGGGCCACCATGGRATGSAGCTGKGTMATSCTC-3′ (SEQ ID NO:22) H36HCF:5′-ATATACTCGCGACAGCTACAGGTGTCCACTCCGAGATCCAGCTGCAGCAGTC-3′ (SEQ IDNO:23) H36HCR: 5′-GACCTGAATTCTAAGGAGACTGTGAGAGTGG-3′ (SEQ ID NO:24)H36LCF: 5′-TTAATTGATATCCAGATGACCCAGTCTCC-3′ (SEQ ID NO:25) H36LCR:TAATCGTTCGAAAAGTGTACTTACGTTTCAGCTCCAGCTTGGTCC (SEQ ID NO:26)

[0094] where in the above SEQ ID NOS: 17 through 26: K is G or T; M is Aor C; R is A or G; S is C or G; V is A, C or G; W is A or T; Y is C orT.

EXAMPLE 2 Binding Activity of Mabs of the Invention

[0095] Mabs of the invention as prepared in Example 1 above wereemployed. The rhTF molecule was expressed in E. coli and purified byimmunoaffinity chromatography in accordance with standard methods (seeHarlow and Lane, supra, Ausubel et al. supra). Mab association (K_(a))and dissociation (K_(d)) constants were determined by ELISA and surfaceplasmon resonance (i.e., BIACore) assays (see e.g., Harlow and Lane,supra; Ausubel et al. supra; Altschuh et al., Biochem., 31:6298 (1992);and the BIAcore method disclosed by Pharmacia Biosensor). For BIACoreassays, rhTF was immobilized on a biosensor chip in accordance with themanufacturer's instructions. Constants for each Mab were determined atfour antibody concentrations (0.125 nM, 0.25 nM, 0.5 nM, and 1 nM).

[0096] Protein concentrations were determined by standard assay (M. M.Bradford, Anal. Biochem., 72:248 (1976)) using Bovine Serum Albumin as astandard and a commercially available dye reagent (Bio-Rad).

[0097]FIG. 2 shows association and disassociation constants for eachanti-rhTF Mab. Mab H36 exhibited the highest association rate(K_(a)=3.1×10⁻¹⁰ M⁻¹) and the lowest disassociation rate(K_(b)=3.2×10⁻¹¹ M) of any of the anti-rhTF Mabs tested.

EXAMPLE 3 FXa-specific Substrate Assay

[0098] In general, the experiments described herein were conducted usingrhTF lipidated with phosphatidycholine (0.07 mg/ml) andphosphatidylserine (0.03 mg/ml) at a 70/30 w/w ratio in 50 mM Tris-HCl,pH 7.5, 0.1% bovine serum albumin (BSA) for 30 minutes at 37° C. A stocksolution of preformed TF:VIIa complex was made by incubating 5 nM of thelipidated rhTF and 5 nM of FVIIa for 30 minutes at 37° C. The TF:VIIacomplex was aliquoted and stored at −70° C. until needed. Purified humanfactors VII, VIIa, and FX were obtained from Enyzme ResearchLaboratories, Inc. The following buffer was used for all FXa and FVIIaassays: 25 mM Hepes-NaOH, 5 mM CaCl₂, 150 mM NaCl, 0.1% BSA, pH 7.5.

[0099] Mabs were screened for capacity to block TF:VIIa-mediatedactivation of FX to FXa. The FX activation was determined in twodiscontinuous steps. In the first step (FX activation), FX conversion toFXa was assayed in the presence of Ca⁺². In the second step (FXaactivity assay), FX activation was quenched by EDTA and the formation ofFXa was determined using a FXa-specific chromogenic substrate (S-2222).The S-2222 and S-2288 (see below) chromogens were obtained fromChromogenix (distributed by Pharmacia Hepar Inc.). FX activation wasconducted in 1.5 ml microfuge tubes by incubating the reaction with 0.08nM TF:VIIa, either pre-incubated with an anti-rhTF antibody or a buffercontrol. The reaction was subsequently incubated for 30 minutes at 37°C., then 30 nM FX was added followed by an additional incubation for 10minutes at 37° C. FXa activity was determined in 96-well titre plates.Twenty microlitres of sample was withdrawn from step one and admixedwith an equal volume of EDTA (500 mM) in each well, followed by additionof 0.144 ml of buffer and 0.016 ml of 5 mM S-2222 substrate. Thereaction was allowed to incubate for an additional 15-30 minutes at 37°C. Reactions were then quenched with 0.05 ml of 50% acetic acid, afterwhich, absorbance at 405 nm was recorded for each reaction. Theinhibition of TF:VIIa activity was calculated from OD_(405nm) values inthe experimental (plus antibody) and control (no antibody) samples. Insome experiments, an anti-hTF antibody, TF/VIIa, and FX were each addedsimultaneously to detect binding competition. FIG. 3 shows that theH36.D2 MAb (in bold) inhibited TF:/VIIa activity toward FX to asignificantly greater extent (95%) than other anti-rHTF Mabs tested.

EXAMPLE 4 FVIIa-Specific Substrate Assay

[0100] Mabs were further screened by an FVIIa specific assay. In thisassay, 5 nM lipidated rhTF was first incubated with buffer (control) or50 nM antibody (experimental) in a 96-well titre plate for 30 minutes at37° C., then admixed with 5 nM purified human FVIIa (V_(T)=0.192 ml),followed by 30 minutes incubation at 37° C. Eight microliters of a 20 mMstock solution of the FVIIa specific substrate S-2288 was then added toeach well (final concentration, 0.8 mM). Subsequently, the reaction wasincubated for one hour at 37° C. Absorbance at 405 nm was then measuredafter quenching with 0.06 ml of 50% acetic acid. Percent inhibition ofTF/VIIa activity was calculated from OD_(405nm) values from theexperimental and control samples.

[0101]FIG. 4 shows the H36 antibody did not significantly block TF/VIIaactivity toward the S-2288 substrate when the antibody was eitherpre-incubated with TF (prior to VIIa addition) or added to TFpre-incubated with VIIa (prior to adding the antibody). This indicatesthat H36 does not interfere with the interaction (binding) between TFand FVIIa, and that H36 also does not inhibit TF:VIIa activity toward apeptide substrate.

EXAMPLE 5 Prothrombin Time (PT) Assay

[0102] Calcified blood plasma will clot within a few seconds afteraddition of thromplastin (TF); a phenomenon called the “prothrombintime” (PT). A prolonged PT is typically a useful indicator ofanti-coagulation activity (see e.g., Gilman et al. supra).

[0103] The H36.D2 antibody was investigated for capacity to affect PTaccording to standard methods using commercially available human plasma(Ci-Trol Control, Level I obtained from Baxter Diagnostics Inc.). Clotreactions were initiated by addition of lipidated rhTF in the presenceof Ca⁺⁺. Clot time was monitored by an automated coagulation timer (MLAElectra 800). PT assays were initiated by injecting 0.2 ml of lipidatedrhTF (in a buffer of 50 mM Tris-HCl, pH 7.5, containing 0.1% BSA, 14.6mM CaCl_(2.) 0.07 mg/ml of phosphatidylcholine, and 0.03 mg/ml ofphosphatidylserine) into plastic twin-well cuvettes. The cuvettes eachcontained 0.1 ml of the plasma preincubated with either 0.01 ml ofbuffer (control sample) or antibody (experimental sample) for 1-2minutes. The inhibition of TF-mediated coagulation by the H36.D2antibody was calculated using a TF standard curve in which the log [TF]was plotted against log clot time.

[0104]FIG. 5 shows the H36.D2 antibody substantially inhibitsTF-initiated coagulation in human plasma. The H36.D2 antibody increasedPT times significantly, showing that the antibody is an effectiveinhibitor of TF-initiated coagulation (up to approximately 99%inhibition).

EXAMPLE 6 FX and the H36.D2 Antibody Compete For Binding to the TF:VIIaComplex

[0105] Competition experiments were conducted between TF/VIIa, FX andthe H36.D2 antibody. FIG. 6A illustrates the results of an experiment inwhich a preformed TF/VIIa complex (0.08 nM) was pre-incubated at 37° C.for 30 minutes in buffer including 0.02 nM, 0.04 nM, 0.08 nM and 0.16 nMof the H36.D2 monoclonal antibody, respectively. FX (30 nM) was thenadded to the TF/VIIa and H36.D2 antibody mixture and the mixture allowedto incubate for an additional 10 minutes at 37° C. FX activation wasquenched with EDTA as described previously. The FXa produced thereby wasdetermined by the FXa-specific assay described in Example 3, above.

[0106]FIG. 6B shows the results of an experiment conducted along thelines just-described, except that the H36.D2 antibody, pre-formedTF:VIIa, and FX were added simultaneously to start the FX activationassay.

[0107] The data set forth in FIGS. 6A and 6B show that the H36.D2antibody and FX compete for binding to the pre-formed TF/VIIa complex.

EXAMPLE 7 Inhibition of TF Activity in Cell Culture

[0108] J-82 is a human bladder carcinoma cell line (available from theATCC) which abundantly expresses native human TF as a cell surfaceprotein. To see if the H36.D2 antibody could prevent FX from binding tonative TF displayed on the cell surface, a J-82 FX activation assay wasconducted in microtitre plates in the presence of FVII (see D. S. Fairet al., J. Biol. Chem., 262:11692 (1987)). To each well, 2×10⁵ cells wasadded and incubated with either 50 ng FVII, buffer (control sample) orthe anti-TF antibody (experimental sample) for 2 hours at 37° C.Afterwards, each well was gently washed with buffer and 0.3 ml of FX(0.05 mg/ml) was added to each well for 30 minutes at room temperature.In some cases, the antibody was added at the same time as FX to detectbinding competition for the native TF. Thereafter, 0.05 ml aliquots wereremoved and added to new wells in a 96-well titre plate containing 0.025ml of 100 mM EDTA. FXa activity was determined by the FXa-specific assayas described in Example 3, above. Inhibition of TF activity on thesurface of the J-82 cells was calculated from the OD_(405nm) in theabsence (control sample) and presence of antibody (experimental sample).

[0109]FIG. 7 shows that the H36.D2 antibody bound native TF expressed onJ-82 cell membranes and inhibited TF-mediated activation of FX. Theseresults indicate that the antibody competes with FX for binding tonative TF displayed on the cell surface. Taken with the data of Example8, below, the results also show that the H36.D2 antibody can bind aconformational epitope on native TF in a cell membrane.

EXAMPLE 8 Specific Binding of the H36.D2 Antibody to Native rhTF

[0110] Evaluation of H36.D2 binding to native and non-native rhTF wasperformed by a simplified dot blot assay. Specifically, rhTF was dilutedto 30 μg/ml in each of the following three buffers: 10 mM Tris-HCl, pH8.0; 10 mM Tris-HCl, pH 8.0 and 8 M urea; and 10 mM Tris-HCl, pH 8.0, 8M urea and 5 mM dithiothreitol. Incubation in the Tris buffer maintainsrhTF in native form, whereas treatment with 8M urea and 5 nMdithiothreitol produces non-native (denatured) rhTF. Each sample wasincubated for 24 hours at room temperature. After the incubation, aMillipore Immobilon (7×7 cm section) membrane was pre-wetted withmethanol, followed by 25 mM Tris, pH 10.4, including 20% methanol. Afterthe membranes were air-dried, approximately 0.5 μl, 1 μl, and 2 μl ofeach sample (30 μg/ml) was applied to the membrane and air-dried. Afterblocking the membrane by PBS containing 5% (w/v) skim milk and 5% (v/v)NP-40, the membrane was probed with H36.D2 antibody, followed byincubation with a goat anti-mouse IgG peroxidase conjugate (obtainedfrom Jackson ImmunoResearch Laboratories, Inc.). After incubation withECL Western Blotting reagents in accordance with the manufacturer'sinstructions (Amersham), the membrane was wrapped with plastic film(Saran Wrap) and exposed to X-ray film for various times.

[0111]FIG. 8A shows that the H36.D2 Mab binds a conformational epitopeon native TF in the presence of Tris buffer or Tris buffer with 8M urea(lanes 1 and 2). The autoradiogram was exposed for 40 seconds. However,when the native TF was denatured with 8M urea and 5mM DTT, H36.D2binding was significantly reduced or eliminated (lane 3). FIG. 8B showsan over-exposed autoradiogram showing residual binding of the H36.D2antibody to non-native (i.e., denatured) rhTF. The over-exposure was forapproximately 120 seconds. Treatment with 8M urea alone probablyresulted in only partial denaturation of the native rhTF since the twodisulfide bonds in TF are not reduced. It is also possible that thepartially denatured TF may refold back to native confirmation duringlater blotting process when urea is removed. These results also clearlydistinguish preferred antibodies of the invention which do not binddenatured TF from previously reported antibodies which do notselectively bind to a conformational epitope and bind to denatured TF(see U.S. Pat. No. 5,437,864 where in FIG. 18 Western Blot analysisshows binding to TF denatured by SDS).

[0112] The invention has been described in detail with reference topreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the disclosure, may makemodification and improvements within the spirit and scope of theinvention.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:26 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GACATTCAGA TGACCCAGTCTCCTGCCTCC CAGTCTGCAT CTCTGGGAGA AAGTGTCACC 60 ATCACATGCC TGGCAAGTCAGACCATTGAT ACATGGTTAG CATGGTATCA GCAGAAACCA 120 GGGAAATCTC CTCAGCTCCTGATTTATGCT GCCACCAACT TGGCAGATGG GGTCCCATCA 180 AGGTTCAGTG GCAGTGGATCTGGCACAAAA TTTTCTTTCA AGATCAGCAG CCTACAGGCT 240 GAAGATTTTG TAAATTATTACTGTCAACAA GTTTACAGTT CTCCATTCAC GTTCGGTGCT 300 GGGACCAAGC TGGAGCTGAA A321 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 107 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Asp Ile Gln Met Thr Gln Ser ProAla Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr CysLeu Ala Ser Gln Thr Ile Asp Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln LysPro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Asn Leu AlaAsp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Lys PheSer Phe Lys Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Asn TyrTyr Cys Gln Gln Val Tyr Ser Ser Pro Phe 85 90 95 Thr Phe Gly Ala Gly ThrLys Leu Glu Leu Lys 100 105 (2) INFORMATION FOR SEQ ID NO: 3: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 351 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:GAGATCCAGC TGCAGCAGTC TGGACCTGAG CTGGTGAAGC CTGGGGCTTC AGTGCAGGTA 60TCCTGCAAGA CTTCTGGTTA CTCATTCACT GACTACAACG TGTACTGGGT GAGGCAGAGC 120CATGGAAAGA GCCTTGAGTG GATTGGATAT ATTGATCCTT ACAATGGTAT TACTATCTAC 180GACCAGAACT TCAAGGGCAA GGCCACATTG ACTGTTGACA AGTCTTCCAC CACAGCCTTC 240ATGCATCTCA ACAGCCTGAC ATCTGACGAC TCTGCAGTTT ATTTCTGTGC AAGAGATGTG 300ACTACGGCCC TTGACTTCTG GGGCCAAGGC ACCACTCTCA CAGTCTCCTC A 351 (2)INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:117 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4: Glu Ile Gln Leu Gln Gln Ser Gly ProGlu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Val Ser Cys Lys ThrXaa Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Tyr Trp Val Arg Gln SerHis Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn GlyIle Thr Ile Tyr Asp Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr ValAsp Lys Ser Ser Thr Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu ThrSer Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Val Thr Thr AlaLeu Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5: Leu Ala Ser Gln Thr Ile Asp 1 5 (2)INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 6: Ala Ala Thr Asn Leu Ala Asp 1 5 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 7: Gln Gln Val Tyr Ser Ser Pro Phe Thr1 5 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8: Thr Asp Tyr Asn Val Tyr 1 5 (2)INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 9: Tyr Ile Asp Pro Tyr Asn Gly Ile ThrIle Tyr Asp Gln Asn Phe Lys 1 5 10 15 Gly (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENTTYPE: N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 10: Asp Val Thr Thr Ala Leu Asp Phe 1 5 (2) INFORMATION FOR SEQ IDNO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 11: CTGGCAAGTC AGACCATTGA T 21 (2) INFORMATIONFOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 12: GCTGCCACCA ACTTGGCAGA T 21 (2) INFORMATIONFOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 13: CAACAAGTTT ACAGTTCTCC ATTCACGT 28 (2)INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACTGACTACA ACGTGTAC 18 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 15: TATATTGATC CTTACAATGG TATTACTATCTACGACCAGA ACTTCAAGGG C 51 (2) INFORMATION FOR SEQ ID NO: 16: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GATGTGACTA CGGCCCTTGA CTTC 24 (2) INFORMATION FOR SEQ ID NO: 17: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GCACCTCCAG ATGTTAACTG CTC 23 (2) INFORMATION FOR SEQ ID NO: 18: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GAARTAVCCC TTGACCAGGC 20 (2) INFORMATION FOR SEQ ID NO: 19: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGAGGCGGCG GTTCTGACAT TGTGMTGWCM CARTC 35 (2) INFORMATION FOR SEQ IDNO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 20: ATTTCAGGCC CAGCCGGCCA TGGCCGARGT YCARCTKCARCARYC 45 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CCCGGGCCACCATGKCCCCW RCTCAGYTYC TKG 33 (2) INFORMATION FOR SEQ ID NO: 22: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCCGGGCCAC CATGGRATGS AGCTGKGTMA TSCTC 35 (2) INFORMATION FOR SEQ IDNO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 23: ATATACTCGC GACAGCTACA GGTGTCCACT CCGAGATCCAGCTGCAGCAG TC 52 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GACCTGAATTCTAAGGAGAC TGTGAGAGTG G 31 (2) INFORMATION FOR SEQ ID NO: 25: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: TTAATTGATA TCCAGATGAC CCAGTCTCC 29 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TAATCGTTCG AAAAGTGTAC TTACGTTTCA GCTCCAGCTT GGTCC 45

What is claimed is:
 1. An antibody that binds native human tissue factorand does not substantially bind non-native tissue factor.
 2. An antibodyof claim 1 wherein the antibody has the binding specificity for nativehuman tissue factor about equal to or greater than H36.D2.B7 [ATCCHB-12255].
 3. An antibody having the binding affinity for native humantissue factor about equal to or greater than H36.D2.B7 [ATCC HB-12255].4. An antibody having identifying characteristics of H36.D2.B7 [ATCCHB-12255].
 5. An antibody of claim 1 wherein the antibody is H36.D2.B7[ATCC HB-12255].
 6. An antibody that binds native tissue factor to forma complex whereby factor X binding to the complex is inhibited.
 7. Anantibody of claim 1 wherein the antibody is a monoclonal antibody.
 8. Anantibody of claim 1 that is a chimeric antibody.
 9. An antibody of claim8 that comprises a constant region of human origin.
 10. An antibody ofclaim 1 that is a single chain antibody.
 11. An antibody that comprisesa sequence that has at least about 70 percent sequence identity to SEQID NO:1.
 12. An antibody of claim 11 that comprises a sequencerepresented by SEQ ID NO:2 or SEQ ID NO:4.
 13. An antibody thatcomprises hypervariable regions that have at least 90 percent sequenceidentity to SEQ ID NOS. 5 through 10 inclusive.
 14. An antibody of claim13 wherein the antibody comprises hypervariable regions represented bySEQ ID NOS. 5 through 10 inclusive.
 15. An isolated nucleic acidcomprising a sequence encoding at least a portion of an antibody thatbinds native human tissue factor.
 16. The nucleic acid of claim 15wherein the monoclonal antibody is H36.D2.B7 [ATCC HB-12255].
 17. Thenucleic acid of claim 15 wherein the nucleic acid comprises SEQ ID NO:1or SEQ ID NO:3.
 18. The nucleic acid of claim 15 wherein the nucleicacid comprises a sequence that has at least about 70 percent sequenceidentity to SEQ ID NO:1 or SEQ ID NO:3.
 19. The nucleic acid of claim 15wherein the nucleic acid comprises sequences coding for antibodyhypervariable regions that have at least 90 percent sequence identity toSEQ ID NOS. 5 through 10 inclusive.
 20. A nucleic acid comprising atleast about 100 base pairs and that hybridizes to SEQ ID NO:1 or SEQ IDNO:3 under normal stringency conditions.
 21. A nucleic acid of claim 20wherein the nucleic acid hybridizes to SEQ ID NO:1 or SEQ ID NO:3 underhigh stringency conditions.
 22. A nucleic acid of claim 15 wherein thenucleic acid comprises sequences that have at least 90 percent sequenceidentitity to SEQ ID NOS. 11 through 16 inclusive and code forhypervariable regions.
 23. A recombinant vector comprising the nucleicacid of claim 15, wherein the vector can express at least a portion ofan antibody that binds native human tissue factor.
 24. A host cellcomprising the vector of claim
 23. 25. A method of inhibiting bloodcoagulation in a mammal, comprising administering to the mammal aneffective amount of an antibody capable of specifically binding nativetissue factor and whereby the antibody complexes with native tissuefactor, and factor X binding to the complex is inhibited.
 26. The methodof claim 25 wherein the complex further comprises factor VII/VIIa. 27.The method of claim 25 wherein the mammal is a human.
 28. The method ofclaim 25 wherein the human is suffering from or suspected of having athrombosis.
 29. The method of claim 25 wherein the human is sufferingfrom or susceptible to restenosis associated with an invasive medicalprocedure.
 30. The method of claim 29 wherein the invasive medicalprocedure is angioplasty, endarterectomy, deployment of a stent, use ofcatheter, graft implantation or use of an arteriovenous shunt.
 31. Themethod of claim 25 wherein the human is suffering from a thromboemboliccondition associated with cardiovascular disease, an infectious disease,a neoplastic disease or use of a thrombolytic agent.
 32. The method ofclaim 25 further comprising administering an anti-platelet composition,a thrombolytic composition or an anti-coagulant composition.
 33. Themethod of claim 25 wherein the antibody is H36.D2.B7 [ATCC HB-12255].34. A method of reducing tissue factor levels in a mammal comprising:administering to the mammal a therapeutically effective amount of anantibody capable of binding native tissue factor, the antibody linkedcovalently to a cell toxin or an effector molecule to providecomplement-fixing ability and antibody-dependent cell-mediatedcytotoxicity, the antibody contacting cells expressing tissue factor toreduce tissue factor levels in the mammal.
 35. The method of claim 34wherein the cells expressing tissue factor are cancer cells, immunecells, or endothelial cells.
 36. A method of detecting tissue factor ina biological sample comprising: contacting a biological sample with amonoclonal antibody of claim 1 and analyzing the biological sample andmonoclonal antibody for the presence of tissue factor in the biologicalsample.